profiling of differentially expressed genes in human ... · protein gs (a subunit), leukocyte...

6
Vol. 4, 3045-3050, December 1998 Clinical Cancer Research 3045 3 The abbreviations used are: HPV, human papillomavirus: RT, reverse transcription. Profiling of Differentially Expressed Genes in Human Primary Cervical Cancer by Complementary DNA Expression Array’ Chanseob Shim, Wei Zhang, Chang Hun Rhee, and Je-Ho Lee2 Center for Clinical Research, Samsung Biomedical Research Institute [C. S.], and Department of Obstetrics and Gynecology, College of Medicine, Sung Kyun Kwan University [J-H. L.J, Samsung Medical Center, Seoul I 35-7 10, Korea: and Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center. Houston, Texas 77030 [W. Z., C. H. R.l ABSTRACT The profiling of differentially expressed genes from primary tumor samples using cDNA expression array can reveal new tumor markers as well as target genes for ther- apeutic intervention. Using cDNA expression array technol- ogy, we produced an expression profile of genes that are associated with human cervical cancer. Hybridization of the cDNA blotting membrane (588 genes on a single membrane) was performed with 32P-labeled eDNA probes synthesized from RNA isolated from either normal cervix or cervical cancer. Parallel analyses of the hybridized signals enabled us to profile genes that were differentially expressed in cervical cancer. In each experiment, the extent of hybridization of each gene was evaluated by comparison with the most abun- dant mRNAs in the human cervix. These include myc proto- oncogene, 405 ribosomal protein 519, heat shock proteins, leukosialin S (CD43), integrin otL (CD1 1A), calgranulin (A), and CDK4 inhibitor (p164). No detectable changes were observed in the expression levels of these genes. Several mRNAs, such as those encoding guanine nucleotide-binding protein Gs (a subunit), leukocyte adhesion protein (LFA1- 1), nuclear factor NF45, homeobox protein Hox-Al, and -catenin were detected in increased levels in cervical can- cer. Genes that showed decreased expression in cervical cancer tissue were a group of apoptosis-related proteins, cell adhesion molecules, nuclear transcription factors, and a homeobox protein (Hox7). For example, the expression 1ev- els of Smadi and Hox7 were consistently decreased in all Received 7/7/98: revised 9/14/98; accepted 9/21/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by G7 Strategic Fund Grant 08-01-17, Samsung Biomedical Research Institute Fund Grant 1-95-016, Korea Science and Engineering Foundation Grant CRC97-2-l through the Cancer Research Center at Seoul National University. and Ministry of Science and Technology of Korea Grant NG1O1O. 2 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, College of Medicine, Sung Kyun Kwan University, Samsung Medical Center, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, Korea. Phone: 82-2-3410-3510: Fax: 82-2-3410-0044; E-mail: [email protected]. tumor tissues tested. Northern analysis of Smadi and Hox7 RNA in primary cervical tumor tissues and cervical carci- noma cell lines indicated that, in general, the mRNA levels of these genes were decreased in human cervical cancer. The precise relationship between the altered expression of these genes and cervical tumorigenesis is a matter of further investigation. INTRODUCTION It is well understood that HPVs,3 especially the high-risk HPV types 16 and 18 (1), are potent etiological entities in the development of cervical cancer. Viral proteins (E6 and E7) from HPV bind to tumor suppressor genes (p53 and the retinoblas- toma genes), leading to the transformation and immortalization of infected cells (2, 3). Inactivation of tumor suppressor genes, by either genomic mutations or virally induced down-regulation of gene expression, results in the deregulation of cell cycle regulatory proteins and activation of various proto-oncogenes (4). However, HPV infection alone is insufficient to trigger the tumorigenic cascade in human cervical cancer cells (4). Only a small fraction of HPV-infected women developed cervical can- cer, indicating that additional factors and cellular events are required for cervical carcinogenesis. These events may include the altered expression of specific genes, such as those encoding proto-oncogenes, tumor suppressors, cell cycle regulatory pro- teins, intracellular signal transducers, apoptosis-associated pro- teins, DNA synthesis/repair/recombination proteins, transcrip- tion factors, cellular adhesion proteins, molecular chaperon proteins, cell surface receptor proteins, and so on. General profiles for the altered expression of such genes in cervical cancer cells are not available. Differential expression cloning has been used as a means of deciphering changes in gene expression in specific cell popula- tions. A unique mRNA subtraction technique that uses PCR has been developed. The so-called cDNA representational differen- tial analysis enables the detection of unique difference in gene expression between related cells (5). Another widely used PCR- based method for detection of changes in gene expression is differential display (6). Differential display allows the study of expression patterns of multiple genes by comparison of PCR- amplified products in adjacent lanes on sequencing gels. In addition, quantitative global analysis of expressed gene se- quence tags by the recently developed serial analysis of gene expression technique (7) may a provide complete catalogue of specific gene expression patterns in the future. The large-scale sequence data generated by the human genome project will then be added to this global profile of expression patterns (8, 9). However, identification of the biological functions of the many Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 American https://bloodcancerdiscov.aacrjournals.org Downloaded from

Upload: others

Post on 15-Jul-2020

4 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

Vol. 4, 3045-3050, December 1998 Clinical Cancer Research 3045

3 The abbreviations used are: HPV, human papillomavirus: RT, reverse

transcription.

Profiling of Differentially Expressed Genes in Human Primary

Cervical Cancer by Complementary DNA Expression Array’

Chanseob Shim, Wei Zhang, Chang Hun Rhee,

and Je-Ho Lee2

Center for Clinical Research, Samsung Biomedical Research Institute[C. S.], and Department of Obstetrics and Gynecology, College of

Medicine, Sung Kyun Kwan University [J-H. L.J, Samsung Medical

Center, Seoul I 35-7 10, Korea: and Department of Neuro-Oncology,

The University of Texas M. D. Anderson Cancer Center. Houston,Texas 77030 [W. Z., C. H. R.l

ABSTRACT

The profiling of differentially expressed genes fromprimary tumor samples using cDNA expression array can

reveal new tumor markers as well as target genes for ther-

apeutic intervention. Using cDNA expression array technol-

ogy, we produced an expression profile of genes that are

associated with human cervical cancer. Hybridization of thecDNA blotting membrane (588 genes on a single membrane)

was performed with 32P-labeled eDNA probes synthesized

from RNA isolated from either normal cervix or cervicalcancer. Parallel analyses of the hybridized signals enabled usto profile genes that were differentially expressed in cervicalcancer. In each experiment, the extent of hybridization ofeach gene was evaluated by comparison with the most abun-dant mRNAs in the human cervix. These include myc proto-

oncogene, 405 ribosomal protein 519, heat shock proteins,

leukosialin S (CD43), integrin otL (CD1 1A), calgranulin (A),and CDK4 inhibitor (p16�4). No detectable changes were

observed in the expression levels of these genes. Several

mRNAs, such as those encoding guanine nucleotide-binding

protein Gs (a subunit), leukocyte adhesion protein (LFA1-

1�), nuclear factor NF45, homeobox protein Hox-Al, and�-catenin were detected in increased levels in cervical can-

cer. Genes that showed decreased expression in cervicalcancer tissue were a group of apoptosis-related proteins, cell

adhesion molecules, nuclear transcription factors, and a

homeobox protein (Hox7). For example, the expression 1ev-els of Smadi and Hox7 were consistently decreased in all

Received 7/7/98: revised 9/14/98; accepted 9/21/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

I This work was supported by G7 Strategic Fund Grant 08-01-17,

Samsung Biomedical Research Institute Fund Grant 1-95-016, KoreaScience and Engineering Foundation Grant CRC97-2-l through theCancer Research Center at Seoul National University. and Ministry ofScience and Technology of Korea Grant NG1O1O.2 To whom requests for reprints should be addressed, at Department ofObstetrics and Gynecology, College of Medicine, Sung Kyun Kwan

University, Samsung Medical Center, 50 Ilwon-Dong, Kangnam-Ku,

Seoul 135-710, Korea. Phone: 82-2-3410-3510: Fax: 82-2-3410-0044;E-mail: [email protected].

tumor tissues tested. Northern analysis of Smadi and Hox7RNA in primary cervical tumor tissues and cervical carci-

noma cell lines indicated that, in general, the mRNA levels ofthese genes were decreased in human cervical cancer. The

precise relationship between the altered expression of these

genes and cervical tumorigenesis is a matter of further

investigation.

INTRODUCTION

It is well understood that HPVs,3 especially the high-risk

HPV types 16 and 18 (1), are potent etiological entities in the

development of cervical cancer. Viral proteins (E6 and E7) from

HPV bind to tumor suppressor genes (p53 and the retinoblas-

toma genes), leading to the transformation and immortalization

of infected cells (2, 3). Inactivation of tumor suppressor genes,

by either genomic mutations or virally induced down-regulation

of gene expression, results in the deregulation of cell cycle

regulatory proteins and activation of various proto-oncogenes

(4). However, HPV infection alone is insufficient to trigger the

tumorigenic cascade in human cervical cancer cells (4). Only a

small fraction of HPV-infected women developed cervical can-

cer, indicating that additional factors and cellular events are

required for cervical carcinogenesis. These events may include

the altered expression of specific genes, such as those encoding

proto-oncogenes, tumor suppressors, cell cycle regulatory pro-

teins, intracellular signal transducers, apoptosis-associated pro-

teins, DNA synthesis/repair/recombination proteins, transcrip-

tion factors, cellular adhesion proteins, molecular chaperon

proteins, cell surface receptor proteins, and so on. General

profiles for the altered expression of such genes in cervical

cancer cells are not available.

Differential expression cloning has been used as a means of

deciphering changes in gene expression in specific cell popula-

tions. A unique mRNA subtraction technique that uses PCR has

been developed. The so-called cDNA representational differen-

tial analysis enables the detection of unique difference in gene

expression between related cells (5). Another widely used PCR-

based method for detection of changes in gene expression is

differential display (6). Differential display allows the study of

expression patterns of multiple genes by comparison of PCR-

amplified products in adjacent lanes on sequencing gels. In

addition, quantitative global analysis of expressed gene se-

quence tags by the recently developed serial analysis of gene

expression technique (7) may a provide complete catalogue of

specific gene expression patterns in the future. The large-scale

sequence data generated by the human genome project will then

be added to this global profile of expression patterns (8, 9).

However, identification of the biological functions of the many

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

Page 2: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

3046 Differential Gene Expression in Human Cervical Cancer

identified genes falls far behind the rapid expansion of the

expressed gene lists.

To begin to understand how the altered expression of a

specific gene plays an integral role in a selected biological

process, one can use the recently developed, high-throughput

technique called cDNA expression array analysis (10, 11). This

technique allows the large-scale comparison of multiple nucleic

acid sequences in a single hybridization. In this study, we used

cDNA expression array technology to analyze genes that are

differentially expressed in human cervical cancer and to identify

complex genetic alterations involved in tumorigenesis and tu-

mor progression.

MATERIALS AND METHODS

Total RNA Isolation. Cervical cancer tissues were ob-

tamed from patients in the Department of Obstetrics and Gyn-

ecology. Samsung Medical Center (Seoul, Korea). The disease

stage of tissue sample was assigned according to the classifica-

tion proposed by the clinical staging criteria of the International

Federation of Gynecology and Obstetrics. Five pairs of normal

and cervical cancer tissues were used in this study. The stages of

cancer were lb to ha. Histologically. all the biopsies were

squamous cell carcinoma. The tissues were frozen in liquid

nitrogen and then stored at -70#{176}C. Before RNA extraction, a

part of each tissue sample was sliced by paraffin section and

examined by H&E staining. Tissue samples containing >50%

tumor cells were used in this study.

Total RNAs were obtained by extracting tissues in Trizol

(Life Technologies Inc., Gaithersburg, MD) according to the

manufacturer’s instructions. Normal cervical tissues and cervi-

cal cancer tissues (- 100 mg of each) were homogenized in

Thzol solution ( 1 ml) using a Polytron homogenizer (Brinkman,

Littau, Switzerland). Homogenates were incubated for 10 mm

on ice, and then a 0.2 volume of chloroform was added to the

homogenates. After vigorous agitation for 5 mm, the inorganic

phase was separated by centrifugation at 12,000 X g for 20 mm

at 4#{176}C.RNAs were then precipitated in the presence of 1 volume

of isopropanol. RNA pellets were washed with 70% ice-cold

ethanol and then dissolved in RNase-free water. Total RNA

concentration was assessed by absorbency at 260 nm using an

UV spectrophotometer (Pharmacia, Uppsala, Sweden).

cDNA Synthesis and Hybridization. 32P-labeled

cDNAs were synthesized with the use of total RNA from both

normal and tumor tissues by RT in the presence of [a-32P]dCTP.

Briefly, total RNAs (20 p.g each) were denatured at 75#{176}Cfor 10

mm in the presence of 8 pmol of dTISVN (V = A, G, and C;

N = A, G, C, and T) mix. After the denaturation step, cDNAs

were synthesized by incubation at 37#{176}Cfor 1 h in a master mix

(total reaction volume = 40 p.1) containing 3 p.1 of dNTP (500

p.M. without dCTP). 5 p.1 of [a-32P]dCTP (3000 Ci/mmol;

Amersham Life Science, Cleveland, OH), and 1600 units of

Moloney murine leukemia virus reverse transcriptase (Promega,

Madison, WI) in 1 X RT buffer (supplied by Promega). The

reaction was terminated by heating for 10 mm at 75#{176}C, and

unincorporated nucleotides were removed by spin column pu-

rification (Chroma Spin-200: Clontech Laboratories Inc., Palo

Alto, CA). For each reaction, -2 X l0� cpm were incorporated

in the final product. After purification, labeled cDNAs were

denatured by boiling for 5 mm and then hybridized to Atlas

human cDNA array blots in hybridization solution (-2 X 106

cpm/ml; ExpressHyb hybridization solution, Clontech). Mem-

branes were prehybridized at 68#{176}Cfor at least 2 h prior to probe

addition. Hybridization was performed at 68#{176}Cin a rolling

bottle overnight. After the first two washes with 2X SSC [1 X

SSC: 0. 15 M NaCI- 15 msi sodium citrate (pH 7.0)] and 0.1%

SDS at 68#{176}Cfor 20 mm, the membranes were subjected to a

stringent wash with 0.1 X SSC, 0.5% SDS, and 0. 1 ms� EDTA

at 68#{176}C.Membranes were then exposed to X-ray film (Hyper-

film, Amersham) for 1 or 3 days at -70#{176}C. The density of each

signal was determined by Fuji MCID-Bas image analysis sys-

tem (Fuji Film, Tokyo, Japan).

Northern Hybridization. For Northern blot hybridiza-

tion, total RNA (10 p.g) was denatured in the presence of 50%

formamide, 2.2 M formaldehyde, 20 m�i 3-[N-morpholino]pro-

panesulfonic acid, 4 mM sodium acetate, and 0.5 mrvi EDTA at

65#{176}Cfor 10 mm. After electrophoresis in a 1 .2% agarose gel

containing 2.2 M formaldehyde, RNA was transferred onto a

nylon membrane (Nytran, 0.45-p.m pore size; Schleicher and

Schuell, Dassel, Germany) by capillary action under lOX SSPE

[1 X SSPE: 0.18 M NaC1, 10 mrvi Na,HPO4 (pH 7.7), and 1 msi

EDTA]. RNA transfer and loading efficiency was estimated by

staining a separate membrane with 0. 1 % methylene blue. RNA

intactness was estimated by comparing the intensities of the 285

and 185 ribosomal RNA bands. For hybridization, the mem-

brane was washed in 6X SSPE for 5 mm and air dried, and the

RNA was permanently attached to the membrane by UV illu-

mination for 1 mm. Hybridization was performed overnight in a

heat-sealable polyethylene bag containing 40 ml of hybridiza-

tion buffer [5X SSPE (pH 7.4), 5X Denhardt’s solution, 0.5%

SDS, 0.2 mg/ml heat-denatured salmon sperm DNA, and 50%

formamide] and the hybridization probe. The Smadi and Hox7

cDNAs, containing the entire coding sequences, were obtained

by PCR amplification and used for hybridization probes. 32P-

labeled cDNA probes were synthesized using a Rediprime

cDNA synthesis kit (Amersham).

RESULTS

The 32P-labeled cDNAs were synthesized by RT of total

RNAs from normal cervical and cervical tumor tissues and used

to hybridize to the cDNA array blots. After stringent washes and

exposure to X-ray film, general expression profiles of 588 genes

in normal cervical and cervical tumor tissues were obtained

(Fig. 1). By comparing hybridized blots for normal versus tumor

tissues, we identified several genes with tumor-specific changes

in expression pattern. Representative genes are marked (Fig. 1,

arrows). As also shown in Fig. 1, no signals are visible in blank

spots and negative control spots (M13 DNA, X phage DNA, and

pUC18 DNA), indicating that hybridization was highly specific.

For low-copy genes that were not detectable with 2 days of

exposure to X-ray film, exposure time was extended (data not

shown). Expression levels of specific genes, either increased or

decreased, were judged by densitometric scanning of hybridized

signals. The hybridization results obtained from five cervical

cancer patients are summarized in Table 1 . Only genes with

expression levels that were altered by > I .5-fold between nor-

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

Page 3: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

p. � � *#{149}�� � .1�.

,* . �

� . #{149}7#{149}� .9

� #{149}4S.-.�#{149}4S � .4 �s #{149}�.�S5

6

� .. ..

Clinical Cancer Research 3047

A.

B.

�1’

�,. .-�#{149}�‘ .�_.u

Fig. 1 Parallel analysis of gene expression in human cervix and cer-

vical cancer using cDNA expression array. 32P-labeled cDNAs were

synthesized by RT of total RNAs isolated from normal cervix (A) or

cervical cancer tissues (B) and hybridized to the Atlas cDNA array blots.

After stringent washes, the blots were exposed to X-ray film for 2 days.

Arrows, representative cDNA spots that show significant differences in

gene expression. Constitutively expressed genes in human cervix: 1,

Mvc: 2, RPS/9; 3, Cyclin H; 4, RPLO; 5, HSP27: 6, HSP7O; 7, Leuko-

sialin S: 8, Integrin ctL: 9, Mch4; /0, Calgranulin (A); II, Smadi; 12,

Hox7.

mal and cancer samples in more than two of the samples are

included.

Constitutive expression of the most abundant mRNAs in

the human cervix was observed in all experiments and did not

show noticeable changes between normal and tumor tissues.

These genes include, among others, myc proto-oncogene, a

series of ribosomal proteins, heat shock proteins, leukosialin S

(CD43), integrin aL (CD1 lA), and calgranulin (A) (Fig. 1,

marked by numbers). Similar expression patterns for these genes

were obtained in independent hybridizations, thus indicating

that our analysis system is reproducible.

Genes that showed increased expression in cervical cancer

tissues are guanine nucleotide binding protein Gs (a subunit),

leukocyte adhesion protein (LFAl-�3), nuclear factor NF45,

homeobox protein Hox-Al, and f3-catenin (Table 1). In four

cervical cancer samples, Gsa expression was consistently higher

than that in normal cervical tissues.

Genes with decreased expression levels in cervical cancer

tissues include a group of apoptosis-related genes, cell cycle

regulators, cell adhesion molecules, nuclear transcription fac-

tors, and homeobox proteins. Particularly interesting is Smad 1,

known as a transforming growth factor-�3 signaling protein-I

(bsp- 1 ), which showed consistently decreased expression levels

in all cancer samples (Fig. 1, 11). Northern analysis of Smadl

mRNA in primary cervical tumors also showed a decreased

expression level, suggesting that a decrease in expression of

Smadl is a general event observed in cervical cancers (Fig. 2).

In contrast, Smad 1 mRNA was detectable in four cervical car-

cinoma cell lines, HeLa, SiHa, Caski, and HT-3. Hox7 (Msxl;

Fig. 1 , 12) expression was decreased in cervical cancer but

highly expressed in human reproductive organs, including en-

dometrium, myometrium, and cervix (Fig. 2). Human ovary and

placenta did not express Hox7 mRNA (Fig. 2). In primary

cervical tumors and cervical carcinoma cells, no Hox7 mRNA

transcripts were detectable by Northern hybridization.

DISCUSSION

In this study, we profiled differentially expressed genes in

human primary cervical cancer by comparing cervical cancer

tissue mRNA with that of normal cervix using cDNA array

technology. Human cDNA array technology is used to examine

simultaneously the expression of specific genes in an entire

cDNA population on a single blot. Although human genome

projects have generated large-scale sequence data for thousands

of genes, the biological functions of such genes remain to be

deciphered. Prior to understanding the functional significance of

specific gene products, it is necessary to define differential gene

expression profiles by comparing the expression patterns of

different tissue types, developmental stages, and disease states.

Although expression analysis techniques such as RT-PCR and

Northern blot analysis have been widely used in the past, these

studies are time consuming and are only applicable to a re-

stricted number of genes. Thus, a systematic approach for ex-

amining large numbers of genes simultaneously is required.

Recently, tools for parallel analysis of gene expression in the

format of ordered gene arrays have been developed.

The Atlas human cDNA expression array system (Clon-

tech) provides a convenient method for profiling the expression

of 588 human genes in one simple experiment. The analytical

principle of human cDNA expression array is based on reverse

Northern blot hybridization. DNA fragments representing 588

human genes are immobilized in duplicate onto a nylon mem-

brane. Each cDNA fragment is 200-500 bp long and is selected

as a unique sequence without a poly(A) tail, repetitive elements,

or highly homologous sequences to minimize cross-hybridiza-

tion and nonspecific binding of a cDNA probe. 32P-labeled

cDNA probes generated by RT of total RNA samples are then

hybridized to an array blot. After a high-stringency wash, the

hybridization pattern can be analyzed by autoradiography.

Among the genes showing differential expression in cervi-

cal cancer, as compared with the normal cervix, were some

previously implicated in tumorigenesis. For example, it is well

known that abnormal G protein signaling, resulting either from

altered gene expression or mutations in the protein coding

region, plays a pathogenetic role in several human diseases (12).

In our analysis, Gsa showed increased expression in four of five

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

Page 4: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

Position#{176} Name of proteinlgene

d3k

d#{244}g

d6i

e6n

e7m

a4m

b5n

c2e

c4g

c4j

c5c

c7b

c7g

d5i

e7f

e7h

Oh

f5gf 7)

(I A gene list is available at Clontech’s world wide web site (http://www.clontech.comJclontechIAPR97UPD/Atlaslist.html).S �,_, I .5-fold or more increased/decreased: ND, not determined (because of high background or not expressed): NC. not changed.

3048 Differential Gene Expression in Human Cervical Cancer

Table I Summary of differently expressed genes in human cervical cancer

Case”

Guanine nucleotide-binding protein S Gsa

Nuclear factor NF45

Homeobox protein Hox-A I

Leukocyte adhesion protein LFA-I�3

�3-CateninMothers against DPP protein/hsp/, Smadl

Extracellular signal-related protein 2/Erk-2

ApopainJCPP32 isoform a

Ich-2 protease/lCErel-lI

Apoptosis inhibitor IAP/IAP2

WSL-LR, WSL-SI, and WSL-52 proteins

Superoxide dismutase/SOD/ [Cu/Zn]Growth arrest and DNA damage-inducible protein/GADDI53

Homeobox protein/Hox7Integrin 31

lntegrin 134Pleiotrophin

Insulin-like growth factor IA

Retinoic acid-binding protein II

I 2 3 4 5

NC + + + +

NC NC + + +

+ ND ND NC +

+ NC + + ND

+ ND ND + ND

- - ND - ND

- ND ND - -

- ND ND - ND

- ND ND - ND

- ND ND - ND

- ND ND - ND- ND ND - ND

- - ND NC ND

- - ND NC ND

- ND ND NC ND

- - ND NC ND

- ND ND - -

- - NC - NC

Normal cervix Cervical cancer

::�i#{216}�

28S� #{149}�

I. P18S ;,

.-.�

E E Cervical cancer.c .� cell lines

�C-G) � � � � � .� c� :� ����V>�O)Q)I c�l�-�

hSmadi � �

LHox7

‘I.

�by*�*#{149}i�i �GAPDH�[��, ,o �Fig. 2 Northern analysis of Smadl and Hox7 genes. Five cases of normal cervical and cervical cancer tissues were used to determine tumor-

specificity of Smad I and Hox7 expression (left). To examine tissue- and cervical carcinoma cell type-specific expression patterns, RNA was extracted

from the placenta, breast, ovary, endometrium, myometrium, and cervix, as well as human cervical carcinoma cell lines, including HeLa, SiHa, Caski,

and HT-3 (right). RNA (10 p.g) was analyzed on a 1.2% denaturing agarose gel and transferred onto a nylon membrane. 32P-labeled cDNA probes

for Smadl and Hox7 were hybridized to the RNA-blotted membranes. After stringent washes, membranes were exposed to X-ray film overnight at

-70#{176}C. For an RNA loading control, the same membranes were rehybridized with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)cDNA.

cases of cervical cancer samples (Table I ), indicating that acti- that lack Smad2 and Smad4 acquire resistance to TGF-13-medi-

vation of Gsa gene expression may be involved in the devel- ated growth inhibition, resulting in cancer progression (14).

opment of cervical cancer. In addition, the Smad proteins, These results indicate that abnormal Smad gene function may

known as downstream effectors of TGF-13-induced signaling, account for the widespread resistance to the growth inhibitory

have been suggested as putative tumor suppressors ( 13). Cells cytokine TGF-13 in human cancers ( 15 ). Smad 1 is a cellular

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

Page 5: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

Clinical Cancer Research 3049

mediator of bone morphogenic protein-induced signal transduc-

tion (16). Although there is no direct evidence for a tumor

suppressive activity of Smad 1 , our results demonstrate that

Smadl may play a role in cervical carcinogenesis.

The Hox7 gene, also known as Msxl, a homeobox-contain-

ing gene in humans ( 17), was also shown to be decreased in

cervical cancer tissues. Although the biological significance of

this finding needs further study, our result shows that altered

expression of the Hox7 gene may be related to cervical cancer

development. Recently, Marazzi et a!. (18) observed that Msx2,

a closely related homologue of Msx 1 , mediates the programmed

cell death pathway induced by bone morphogenic protein-4 in

P19 embryonal carcinoma cells. In addition, the close spatial

correlation of Msxl and Msx2 gene expression with the site of

programmed cell death has provided indirect evidence for Msx

gene function in cellular proliferation and differentiation (19).

Our Northern analyses showed that Hox7 niRNA was expressed

predominantly in reproductive organs that show cyclic changes

in growth and differentiation according to the reproductive

stages (that is, endometrium, myometrium, and cervix; Fig. 2).

This result is consistent with a report showing that, in adult

mouse, Hox7 mRNA was only detectable in the MOllerian

epithelium of the uterus, cervix, and vagina (20). In primary

cervical cancers and cervical carcinoma cell lines (HeLa, SiHa,

Caski, and HT-3), no Hox7 mRNA was detectable by Northern

hybridization, indicating that a close correlation exists between

Hox7 expression and tumorigenesis. Deregulation of homeobox

proteins Hox and Pax has been reported in various human

cancers (21-25). However, the mechanisms by which ho-

meobox gene products regulate tumeric growth are largely un-

known.

Considering that cell-surface adhesion molecules are in-

volved in the regulation of cell growth and movement, their

abnormal functions may lead to cancer development (26). Inte-

grins are cell surface heterodimeric proteins that mediate cell-

cell and cell-extracellular matrix interactions, and deregulation

of integrin gene expression is commonly observed in various

tumors (27, 28). Our detection of decreased levels ofthe integrin

subunits 131 and 134 in cervical cancer (Table 1 ) is consistent

with previous results (29-3 1). Disruption of the programmed

cell death cascade and inappropriate cell cycle regulation are

likely involved in abnormal cellular growth and tumorigenesis

(32, 33). Thus, it is not strange that cell death-related genes

involved in the apoptotic signal cascade are differentially ex-

pressed in cervical cancer.

In conclusion, our study demonstrates the use of the cDNA

array technique in monitoring the overall profile of gene expres-

sion in cervical tumorigenesis. The profiling of additional pri-

mary tumor samples will help to reveal genes that are commonly

expressed in this type of tumor. Information from these studies

may be useful in the development of therapeutic drugs for the

treatment of cervical cancer.

REFERENCES

1. Lorincz, A. T., Temple, G. F., Kurman, R. J., Jenson, A. B., and

Lancaster, W. D. Oncogenic association of specific human papillomavirus types with cervical neoplasia. J. Int. Cancer Inst., 79: 67 1-677,

1987.

2. Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., andHowley. P. M. The E6 oncoprotein encoded by human papillomavirustypes 16 and 18 promotes the degradation of p53. Cell, 63: 1 129-1 136,

1990.

3. Scheffner, M., Munger, K., Huibregtse, J. M., and Howley, P. M.

Targeted degradation of the retinoblastoma protein by human papillo-

mavirus E7-E6 fusion proteins. EMBO J., II: 2425-2431, 1992.

4. Park, T-W., Fujiwara, H., and Wright. T. C. Molecular biology of

cervical cancer and its precursors. Cancer (Phila.). 76 (Suppl.): 1902-

1913, 1995.

5. Hubank, M., and Schatz, D. G. Identifying differences in mRNA

expression by representational difference analysis. Nucleic Acid Res..

22: 5640-5648, 1994.

6. Liang, P., and Pardee, A. B. Differential display of eukaryoticmessenger RNA by means of the polymerase chain reaction. Science

(Washington DC), 257: 967-97 1 , 1992.

7. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W.

Serial analysis of gene expression. Science (Washington DC), 270:

484-487, 1995.

8. Hiller, L. D., Lennon, G., Becker, M., Bonaldo. M. F., and Chiapelli,

B. Generation and analysis of 280,000 human expressed sequence tags.Genome Res., 6: 807-828, 1996.

9. Aaronson, J. S., Eckman, B., and Belvins, R. A. Toward the devel-opment of a gene index to the human genome: an assessment of the

nature of high-throughput EST sequence data. Genome Res., 6: 829-845. 1996.

10. DeRisi, J., Penland. L., Brown, P. 0., Bittner, M. L., Meltzer, P. S.,

Ray, M., Chen, Y., Su, Y. A., and Trent, J. M. Use of a cDNAmicroarray to analyse gene expression patterns in human cancer. Nat.

Genet., 14: 457-460, 1996.

I 1. Heller, R. A.. Schena, M., Chai, A., Shalon, D., Bedilion, T.,

Gilmor, J., Wooley, D. E., and Davis, R. W. Discovery and analysis of

inflammatory disease-related genes using cDNA microarrays. Proc.

NatI. Acad. Sci. USA, 94: 2150-2155, 1997.

12. Weinstein, L. S., and Shenker, A. G protein mutations in humandisease. Clin. Biochem.. 26: 333-338, 1993.

13. Lechleider. R. J., de Caestecker, M. P.. Dehejia, A., Polymeropou-

los, M. H., and Roberts, A. B. Serine phosphorylation, chromosomal

localization, and transforming growth factor-13 signal transduction by

human bsp-l. J. Biol. Chem., 271: 17617-17620, 1996.

14. Nakao, A.. Roijer, E., Imamura, T., Souchelnytskyi. S.. Stenman.

G., Heldin, C. H., and Dijke, P. Identification of Smad2, a human

Mad-related protein in the transforming growth factor 13 signaling path-

way. J. Biol. Chem., 272: 2896-2900, 1997.

15. Riggins, G. J., Kinzler, K. W., Vogelstein, B., and Thiagalingam, S.Frequency of Smad gene mutations in human cancers. Cancer Res., 57:

2578-2580. 1997.

16. Liu, F., Hata, A.. Baker. J. C., Doody. J., Carcamo, J.. Harland.R. M., and Massagu#{233},J. Human Smadl, a mediator of BMP signals, is

a transcription activator. Nature (Lond.), 381: 620-623, 1996.

17. Hewitt, J. E., Clark, L. N., Ivens, A., and Williamson, R. Structure

and sequence of the human homeobox gene HOX7. Genomics, II:

670-678, 1991.

18. Marazzi, G., Wang, Y., and Sassoon, D. Msx2 is a transcriptional

regulator in the BMP4-mediated programmed cell death pathway. Dev.

Biol., 186: 127-138, 1997.

19. Davidson, D. The function and evolution of MSX genes: pointers

and paradoxes. Trends Genet.. 11: 405-41 1, 1995.

20. Pavlova, A., Boutin, E., Cunha, G., and Sassoon, S. Msxl (Hox-7.l)

in the adult mouse uterus: cellular interactions underlying regulation of

expression. Development (Camb.), 120: 335-346, 1994.

21. Barba, P., Magli. M. C., Tiberio, C., and Cillo, C. HOX geneexpression in human cancers. Adv. Exp. Med. Biol., 348: 45-57. 1993.

22. Cillo. C. HOX genes in human cancers. Invasion Metastasis, 14:

38-49, 1994.

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

Page 6: Profiling of Differentially Expressed Genes in Human ... · protein Gs (a subunit), leukocyte adhesion protein (LFA1-1), nuclear factor NF45, homeobox protein Hox-Al, and-catenin

3050 Differential Gene Expression in Human Cervical Cancer

23. Stuart, E. T., and Gruss, P. PAX genes: what’s new in developmen-

tal biology and cancer! Hum. Mol. Genet., 4: 1717-1720, 1995.

24. Stuart, E. T., Yokota, Y., and Gruss, P. PAX and HOX in neoplasia.

Adv. Genet., 33: 255-274, 1995.

25. Anbazhagan, R., and Raman, V. Homeobox genes: molecular linkbetween congenital anomalies and cancer. Eur. J. Cancer, 33: 635-637,

1997.

26. Giancotti, F. G. Integrin signaling: specificity and control of cell

survival and cell cycle progression. Curr. Opin. Cell Biol., 9: 691-700,

1997.

27. Juliano, R. L., and Varner, J. A. Adhesion molecules in cancer: therole of integrins. Curr. Opin. Cell Biol., 5: 812-818, 1993.

28. Varner, J. A., and Cheresh, D. A. Integrins and cancer. Curr. Opin.Cell Biol., 8: 724-730, 1996.

29. Aplin, J. D., Dawson, S., and Seif, M. W. Abnormal expression of

integnn a6134 in cervical intraepithelial neoplasia. Br. J. Cancer, 74:

240-245 1996.

30. Hodivala, K. J., Pci, X. F., Liu, Q. Y., Jones, P. H., Rytina, E. R.,Gilbert, C., Singer, A., and Wart, F. M. Integrin expression and functionin HPV 16-immortalised human keratinocytes in the presence or ab-

sence of v-Ha-ras. Comparison with cervical intraepithelial neoplasia.Oncogene. 9: 943-948, 1994.

31. Carico, E., French, D., Bucci. B., Falcioni, R., Vecchione, A., and

Mariani-Costantini, R. Integrin 134 expression in the neoplastic progres-sion of cervical epithelium. Gynecol. Oncol., 49: 61-66, 1993.

32. Metcalfe, A., and Streuli, C. Epithelial apoptosis. Bioessays, 19:

711-720, 1997.

33. Wyllie, A. H. Apoptosis and carcinogenesis. Eur. J. Cell Biol., 73:

189-197, 1997.

Association for Cancer Research. by guest on August 28, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from